Utilizing the three carbonic anhydrase (CA) isozymes (CA I, CA II, CA III) as a model multilocus enzyme system, we propose to study the molecular evolution of isozymes derived from a common ancestral gene, as well as the mechanisms which regulate their expression in human and other vertebrate cells. In particular, we are directing our research toward: 1) the evolutionary origins of the human CA isozymes, 2) the effect of mutations and evolutionary change on the regulation and function of the different CA isozymes, and 3) the organization and structure of CA genes in mammalian cells. We plan to continue our studies on the amino acid sequences of several CA isozymes purified from a variety of mammalian and non-mammalian sources, and use this information to study the comparative rates of evolution of the CA isozymes since their origin by gene duplication from a common ancestral gene. We will also attempt to chemically characterize several recently discovered variants of human red cell CA I and CA II. Because of the rather unusual (compared to the CA I and II isozymes) kinetic and distributional characteristics of the recently discovered CA III isozymes of mammalian muscle and liver, we wish to expand our preliminary studies on the modificatton of residues unique to the active site of the CA III molecules purified from human as well as selected non-mammalian sources. Through such studies, we hope to understand more about the "true" physiological roles of the CA III isozymes.